Evidence of Orientia spp. Endemicity among Severe Infectious Disease Cohorts, Uganda

At 3 severe infection cohort sites in Uganda, Orientia seropositivity was common. We identified 4 seroconversion cases and 1 PCR-positive case. These results provide serologic and molecular support for Orientia spp. circulating in sub-Saharan Africa, possibly expanding its endemic range. Orientia infections could cause severe illness and hospitalizations in this region.

for enrollment.Patients were not eligible if they were deemed too ill to participate with an imminently terminal comorbidity or if they presented with severe anemia (hemoglobin <7 g/dL).
Due to inflammation biomarker objectives, participants were excluded with known immunocompromising conditions, including drug induced immunosuppression, anatomic or functional asplenia, recent chemotherapy, pregnancy and <6 weeks postpartum females, but participants with HIV were eligible.FPRRH serves as a health facility to eight districts in western Uganda.

Sepsis and AFI Cohort
If there was clinical suspicion, PCR testing for tuberculosis was performed using expectorated sputum (Xpert MTB/RIF Ultra, Cepheid, Sunnyvale, CA, USA) and participants with HIV had a urine lipoarabinomman (LAM test).Whole blood was run on the FilmArray Global Fever panel for 19 non-rickettsial pathogen targets (3).

Seroconversion Criteria
To validate the findings of seropositivity, we also read Gilliam strain IFA slides (BIOCELL Diagnostics Inc., Baltimore, MD, USA) for a portion (n = 50), including those that seroconverted and had a >1:256 titer (n = 13) and also 37 (12%) randomly selected samples.We used seroconversion criteria to decrease the risk of a false Karp IFA seroconversion.First, we included seroconversions defined as a >4-fold increase in titer from acute to convalescent sera resulting in a convalescent titer of at least 512, excluding n = 4 at 256 (the upper interquartile range [IQR] of the Orientia IgG titers).Second, while serum cross-reactivity with spotted fever group rickettsia (SFGR) or typhus group rickettsia (TGR) has not been widely reported (4), we excluded participants with convalescent SFGR (n = 7) or TGR (n = 2) IFA IgG titers that were greater than or equal to Orientia spp.(SFGR Rickettsia conorii Molish 7 strain, TGR Rickettsia typhi Wilmington strain; BIOCELL Diagnostics Inc, Baltimore, MD, USA).Third, we excluded those with severe symptoms present for >14 days at time of enrollment (n = 2).

Western Blot and Dot Blot
For the Western blot and dot blot, we used the TSA 56-kDa Orientia protein (5) to determine existence of Orientia spp.antibodies from the serum sample.The recombinant protein was received frozen after preparation using the referenced procedures (5) but was dissolved in 8 M urea.The protein was dialyzed to remove urea using Amicon Ultra 0.5 mL centrifugal filter units (Merck), following established procedures.Centrifugation occurred at 1,000 to 2,000 g for ten minutes and was followed by a wash step with phosphate-buffered saline (PBS, pH 7.4) and an additional centrifugation.The dialyzed protein was carefully collected from the filter units and stored in PBS.Positive controls included Orientia tsutsugamushi IgG-positive serum and a healthy unexposed human serum negative control.
For the Western blot, recombinant TSA56 protein was prepared in two concentrations, 2 µg and 5 in NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent (Invitrogen, Thermo Fisher Scientific).The samples underwent denaturation at 70°C for 10 minutes using a heat block (Thermo Fisher Scientific) and were loaded onto a NuPAGE 10% Bis-Tris gel (Invitrogen, Thermo Fisher Scientific).Electrophoresis was conducted at 200V.The proteins were transferred to a membrane using the Trans-Blot Turbo Transfer System (Bio-Rad).
Transfer efficiency was assessed by Ponceau S staining (Sigma-Aldrich).The membrane was blocked with 5% non-fat dry milk in PBST (PBS + 0.1% Tween-20) and then incubated with human serum samples diluted 1:100,000, including a positive control serum containing scrub typhus IgG and a negative control of normal human serum.The membrane was treated with HRP-conjugated anti-human IgG (Invitrogen), diluted to 1:2,000.Subsequently, to assess protein purity, the TSA56 protein weight was estimated using ImageJ software (6).After log10 transformation to fit a standard curve, the weight of the TSA56 protein was estimated to be 59.8 kDa and, accordingly, within range of anticipated measurement error of 56 kDa.
For the dot plot, we used a nitrocellulose membrane.we prepared a nitrocellulose membrane (Bio-Rad) and applied TSA56 recombinant protein (2) and bovine serum albumin (BSA), the latter serving as a negative control to assess antibody specificity.To ensure nonspecific binding was identified, Rickettsia parkeri (Rp) protein was used as an unrelated antigen.
The assay incorporated positive controls consisting of scrub typhus IgG serum and negative controls of normal human serum.Blocking was conducted using a buffer of 5% non-fat dry milk in PBST (PBS + 0.1% Tween-20) to prevent non-specific binding.The secondary antibody used was HRP-conjugated anti-human IgG (Invitrogen). 1 µg of TSA56 protein, BSA and Rp were spotted onto the membrane, followed by air drying.Serum samples were diluted ranging from 1:100 to 1:100,000 in blocking buffer and incubated on the membrane followed by a 1-hour room temperature incubation.Signal was detected for both the dot plot and the western blot using iBright FL1000 (Thermo Fisher Scientific).